Differential survival of cytotoxic T cells and memory cell precursors

It is widely assumed that the development of memory CD8 T cells requires the escape of CTLs from programmed cell death. We show in this study that although serine protease inhibitor 6 (Spi6) is required to protect clonal bursts of CTLs from granzyme B-induced programmed cell death, it is not required for the development of memory cells. This conclusion is reached because memory cell precursors down-regulate both Spi6 and granzyme B, unlike CTLs, and they do not require Spi6 for survival. These findings suggest that memory CD8 T cells are derived from progenitors that are refractory to self-inflicted damage, rather than derived from fully differentiated CTLs.     

Antibodies to T cell receptors and to histocompatibility antigens

The injection of 10⁶ parental strain spleen cells into adult F1 hybrid mice resulted in the formation of two serum activities: anti-receptor antibodies and alloantibodies. To demonstrate the respective roles of T and B cells in the elicitation of these serum activities, parental strain lymphoid cell suspensions containing varying proportions of T and B cells, or consisting only of T or B cells, were employed as inocula for F1 mice. The results indicated that the injection of pure T cells led to the formation of antireceptor antibodies only, while the injection of pure B cells resulted exclusively in formation of alloantibodies, suggesting that anti-receptor antibodies were structures elicited by T cell receptors. Alloantibodies appear to be formed as a consequence of the interaction of B cell receptors with alloantigen.     

T cell tolerization in vitro: modulation of helper and suppressor cell activities

This paper analyzes the conditions for in vitro tolerization of purified whole T cell populations and the consequences on helper and suppressor T cell functions. Highly purified splenic T cells from adult DBA/2 mice were incubated in vitro for 24 hr with high doses of trinitrophenyl coupled to human gamma-globulins (TNP-HGG). A profound inhibition of the TNP-specific helper function of these T lymphocytes was observed in a cooperative culture with normal purified splenic B cells and TNP-SRBC as antigen. This state of specific unresponsiveness was maintained after trypsin treatment of the cells, at the end of the 24-hr incubation with the tolerogen. We checked that this procedure removed the vast majority of F23.1 T cell receptor determinants from the cells. This result indicates that T cell receptors for antigen were not merely blocked by the tolerogen. In addition, B cells preincubated with tolerized T cells for 24 hr remained as responsive to TNP as B cells mixed with normal T cells in similar conditions. This demonstrates that the decreased response is not the result of secondary B cell tolerization. In addition, anti-Ia monoclonal antibodies were shown to block the induction of tolerance. We also showed that tolerized T cells significantly decreased the anti-TNP response of normal T and B cells in vitro, whereas the anti-SRBC response in the same cultures was unaffected. When tolerized T cells were separated into Lyt-2- and Lyt-2+ cells, it was found that tolerized Lyt-2- cells had lost about 75% of their helper activity and that Lyt-2+ cells suppressed 70% of the response of a normal T and B cell culture. Thus, in vitro induction of T cell tolerance results in a specific T cell unresponsiveness which is due to both helper T cell inactivation and induction of specific suppressor T cells.     

Cellular interactions in the cytotoxic T lymphocyte response to herpes simplex virus antigens: differential antigen activation requirements for the helper T lymphocyte and cytotoxic T lymphocyte precursors

The role and induction requirements of helper T lymphocyte responses to herpes simplex virus type 1 (HSV-1) was examined. Splenocytes from mice that had been primed in vivo with infectious HSV-1 can be restimulated in vitro with live or partially UV-inactivated HSV-1 to generate high levels of herpes virus-specific cytotoxic T lymphocyte (CTL) activity. By comparison, naive splenocytes or splenocytes taken from mice primed with heat-inactivated HSV-1 failed to generate CTL after in vitro viral stimulation. In addition, infectious HSV-primed splenocytes can be rendered unresponsive to secondary in vitro restimulation by pretreatment with anti-Lyt-1 antiserum plus complement. Spleen cells were taken from mice that had been primed and restimulated in vivo with infectious HSV-1. Two days after the second priming, splenocytes were prepared and irradiated. These cells were capable of assisting in the generation of CTL to varying degrees in all of the above unresponsive populations of cells. The irradiated cells did not produce detectable levels of CTL activity when cultured alone with antigen. Also, if the irradiated splenocytes were treated with anti-Lyt-1 plus complement before their addition to cultures, all restorative activity was ablated. In contrast, irradiated splenocytes from mice that had been primed and restimulated in vivo with either heat-inactivated or UV-inactivated HSV-1 were unable to provide help to naive or helper-depleted cultures. The failure to supply helper activity appears not to involve the preferential activation of suppressor cells, as evidenced by cell mixing experiments and the addition of concentrated, antigen-stimulated spleen cell supernatant fluids to secondary anti-HSV-1 splenocyte cultures. Proliferative assays using interleukin 2- (IL 2) dependent cell lines as a measure of relative helper activity indicated that the inactivated forms of HSV-1 were incapable of effectively enlisting helper activity. These experiments therefore suggest that the observed failure of heat-inactivated or UV-inactivated HSV-1 preparations to induce anti-HSV CTL responses reflects the inability of the HSV-1-specific subset of helper T lymphocytes to recognize these forms of the antigen.     

A requirement for physical linkage between determinants recognized by helper molecules and cytotoxic T cell precursors in the induction of cytotoxic T cell responses

It has long been understood that both antibody and delayed-type hypersensitivity responses are induced through collaborative events in which the determinants recognized by the precursor cells must be physically linked to the determinants recognized by the helper. Although it is clear that the generation of memory cytotoxic T lymphocyte precursors (CTLp) involves linked recognition of determinants, the induction of CTL responses has been viewed as being dependent upon interleukin 2 (IL 2), which could be provided by a helper cell, but independent of requirements for antigen bridging. In this work, we have designed a system that lacks exogenous IL 2 by using as our source of help, antigen-specific helper molecules derived from helper T cells. These soluble helper molecules are uncontaminated by IL 2 and unlike a helper cell, are unable to produce IL 2. Helper molecules specific for chicken red blood cells (Crbc) and for a synthetic polypeptide, poly 18, were tested. Thymocyte responders require a source of help to respond to alloantigens intrinsically expressed on the surface of adherent stimulator cells. To analyze the mechanism whereby the helper molecules acted, we used a system involving recognition of haptenic and carrier determinants that were physically linked by virtue of being located on the same cell surface (intra-structural linkage). Adherent stimulator cells were pulsed with Crbc or poly 18 so that the alloantigens recognized by the thymocyte CTLp (intrinsically expressed class I) were either linked or unlinked to the carrier determinants (Crbc or poly 18) presented by the adherent cells and recognized by the helper molecules. Both types of helper molecule were shown to be antigen-specific in crisscross experiments. The helper molecules specific for Crbc were able to induce the thymocyte CTLp only when both hapten and carrier were present on the same stimulator cell surface. Because we were not able to detect a requirement for H-2-restricted recognition of carrier antigen, this inductive event must be viewed as requiring linked associative recognition of determinants, but being noncognate. In contrast, the helper molecules recognizing poly 18 showed a requirement for both physical linkage of determinants and for H-2 restricted recognition, indicating that the mechanism of induction was cognate in nature. Therefore, we have shown that interactions between CTLp and soluble, antigen-specific, helper cell-derived inductive molecules are similar in nature to those of other T cell precursors and of B cells in the stringent requirement for close physical proximity achieved by linked or cognate recognition of determinants across an antigen bridge.     

Differential susceptibility of leukocyte subsets to cytotoxic T cell killing: implications for HIV immunopathogenesis.

BACKGROUND: Cytotoxic T lymphocytes (CTL) are crucial for the host defense against viral infection. In many cases, this anti-viral immune response contributes to host pathogenesis, through inflammation and tissue destruction. Few studies have explored the relative susceptibility of infected cells to CTL killing, and the range of cell types that may be effectively killed by CTLs in vivo, both of which are key to understanding both immune control of infection and immune-related pathogenesis. METHODS: We developed and optimized a highly sensitive method to quantify the relative susceptibility of leukocyte subsets to CTL-mediated killing. Maximal sensitivity was achieved by uniquely measuring cell death occurring during the assay culture. RESULTS: We found that leukocyte subsets have a wide range of susceptibility to antigen-specific CTL-mediated lysis. Generally, T cells were more susceptible than B or NK cells, with CD4 T cells being more susceptible than CD8 T cells. In all lymphocyte lineages, susceptibility was greater for more differentiated subsets compared with their na?ve counterparts; however, for dendritic cells, immature cells are more susceptible than mature cells. We focused on the susceptibility of T cell subsets, and found that na?ve cells are far more resistant than memory cells, and in particular, CCR5+ or HLA-DR+ memory cells are highly susceptible to CTL-mediated killing. CONCLUSIONS: These results provide an explanation for the observation that certain subsets of CD4 T cells are ablated during chronic HIV infection, and indicate which subsets are most likely to contain the persistent viral reservoir.     

Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens

A model for bone marrow transplantation across minor histocompatibility barriers was developed by using mouse strains that were H-2 identical and mutually non-reactive in MLC. Acute graft-vs-host disease was induced only when donor lymphoid cells were included in the marrow inoculum, in both C57BL/6 recipients of LP cells and BALB/c recipients of B10.D2/nSN cells. GVHD was prevented by treating the lymphoid cells with anti-Thy 1.2 and C before transplantation. Spleen cells from mice with acute GVHD were not directly cytotoxic to recipient strain target cells. However, when spleen cells from mice with GVHD were boosted in vitro to recipient strain stimulator cells they generated a specific anti-recipient cytotoxic response. Spleen cells from mice without GVHD did not generate a cytotoxic response in vitro. The cytotoxic effector cells and their precursors were shown to be T lymphocytes. This model and the in vitro method described may be useful in further studies of the immunobiology of GVHD due to minor histocompatibility antigens and of transplantation tolerance.     

The development of helper T cell precursors in mouse thymus

We have examined the appearance in mouse ontogeny of thymocyte precursors for Ag-specific, MHC-restricted Th. These cells are first detectable at day 18 of fetal life, about 1 day after alpha/beta, TCR-positive cells begin to appear. These early Th precursors are not dependent on the thymus for priming with Ag and MHC, and are L3T4+, Lyt-2-. Thus, these cells already have the phenotype of mature Th. In neonatal F1 animals expressing both IAk and IAb, the appearance of Th precursors restricted by either IAk or IAb is specifically inhibited by treatment of the mice with anti-IAk or anti-IAb antibodies, respectively. These results indicate that cells of mature T cell phenotype and function can arise fairly rapidly from immature, receptor-bearing precursors, once these appear. Moreover the results are in line with those previously obtained in chimeric animal experiments which suggested that specific interaction of TCR on thymocytes with class II alleles in the thymus is required for the subsequent appearance of T cells restricted by those class II alleles.     

Functional differentiation of T cell precursors. I. Parameters of carrier-specific tolerance in murine helper T cell precursors.

Irradiated mice reconstituted with bone marrow from sheep gamma-globulin- (SGG) tolerant syngeneic donors display reduced IgG responsiveness to challenge with trinitrophenylated (TNP)-SGG compared with recipients of normal marrow. This effect is SGG-specific and is due neither to suppressor T cells nor to antigen carryover. "Helper T cell precursor tolerance" can be induced with as little as 40 micrograms tolerogen (SGG). Unlike mature helper T cells, these precursors show both a rapid induction and rapid waning patterns, suggesting a high rate of turnover. Our results imply that marrow helper T cell precursors bear antigen-specific receptors and that the T cell repertoire must be at least partially generated before residence in the thymus.     

Subpopulations of CD8+ cytotoxic T cell precursors collaborate in the absence of conventional CD4+ helper T cells.

Four different subpopulations (Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi) of CD8+ T cells were arbitrarily defined on the basis of differential expression of Ly6C Ag. By combining the processes of electronic cell sorting and automated cell deposition, small numbers of respective CD8+ T cell subpopulations were directly deposited into tissue culture wells in which mitogen-stimulated responses were studied. Anti-CD3-stimulated proliferation and IL-2 production were the strongest by Ly6Cneg/Ly6Clow T cells, moderate for Ly6Cint T cells, and highly deficient for Ly6Chi T cells. The level of IL-2 production for Ly6Cneg CD8+ T cells was comparable to that of conventional CD4+ Th cells. Allogeneic stimulator cells elicited a strong cytotoxic response by Ly6Cneg + low but not Ly6Chi CD8+ T cells in the absence of added lymphokines. When IL-2 was supplied in excess, anti-CD3 induced comparable levels of cell proliferation and cytotoxic activity in Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi CD8+ T cells whereas alloantigen stimulated an approximate fivefold higher cytotoxic response by Ly6Chi than Ly6Cneg + low CD8+ T cells. Stimulation of co-cultures of B10 (CD8b) Ly6Cneg + low and congenic B10.CD8a Ly6Chi CD8+ T cells in the absence of added lymphokines, followed by selective elimination of activated CD8.1+ (CD8.2+) T cells by anti-CD8.1 (anti-CD8.2) + C treatment, allowed the demonstration that help provided by Ly6Cneg + low T cells can be effectively used by both Ly6Cneg + low and Ly6Chi T cells in anti-CD3 and alloantigen induced proliferative and cytotoxic responses, respectively.     

Immunodominance in the graft-vs-host disease T cell response to minor histocompatibility antigens

Immunodominance controls the generation of CTL in the C57BL/6By (B6) anti-BALB.B H-2b-matched strain combination. Despite the potential of responding to numerous individual minor histocompatibility (H) Ag on BALB.B APC, the focus of the CTL response is largely specific for only a limited number of target Ag. These minor H Ag could be distinguished by their differential expression on a panel of target cells from the CXB recombinant inbred strains, the E, G, I, J, and K (all H-2b), which express different composites of the original BALB minor H Ag. A hierarchy was observed in which first-order immunodominant Ag were present on both CXBK and CXBG cells, whereas second-order dominant Ag were found on CXBE, CXBJ, and CXBI cells. To test whether immunodominance also plays a role in the development of lethal graft-vs-host disease (GVHD) directed to multiple minor H Ag, B6 T cells were transplanted along with T cell depleted bone marrow, to irradiated (825 rad) recipients of either the BALB.B or CXB recombinant inbred strains. The results indicate that a hierarchy of immunodominance does exist in GVHD, but it differs from that predicted from the in vitro CTL studies. GVHD was observed in BALB.B, CXBE, CXBI, and CXBJ recipients, but not in CXBG and CXBK recipients. Presensitization of B6 donor mice to CXBG or CXBK splenocytes 3 wk before transplant did not significantly increase the overall GVHD potential in the corresponding CXBG or CXBK recipients. Evidence for second-order immunodominance was provided by the transfer of CXBE T cells and ATBM to irradiated CXBG and BALB.B recipients with resultant, potent GVHD.     

Killing of mouse blastocyst stage embryos by cytotoxic T lymphocytes directed to major histocompatibility complex antigens

A new assay, mixed embryo leukocyte interaction assay, in which the ability of cytotoxic T lymphocytes (CTL) to kill preimplantation mouse embryos could be investigated, is described. CTL were generated both in vitro and in vivo to the H-2b and H-2d haplotypes. The specificity of the CTL was verified by using EL-4 (H-2b) and P815 (H-2d) target cells in a 51Cr-release assay. The cytolytic effect of the CTL on mouse blastocysts was measured by assessing blastocoel retention and inhibition of [3H]thymidine incorporation by the embryos. It was shown that CTL kill blastocyst stage embryos from C57BL/6J (H-2b) and B10.D2 (H-2d) mice with the zona pellucida removed, but not with the zona pellucida intact. These results demonstrate that the H-2 antigens present on mouse blastocysts can be recognized by CTL. It is suggested that one biologic role for the zona pellucida is the prevention of cell-mediated destruction of preimplantation embryos in utero.     

Successful priming and tolerization of T cells to orally administered antigens in B-cell-deficient mice

B cells have been shown to function as APCs capable of inducing both T cell priming and tolerization. Recently, B cells were also revealed to be essential in the organogenesis of Payer's patches (PPs), which have been supposed to play an important role in the initiation of mucosal immune responses. In this study, we examined the roles of B cells in T cell response to orally administrated antigen using B-cell-deficient mice. It was revealed that (1) both a single high dose and repeated low doses of orally administered OVA successfully induced tolerance of T cells in B-cell-deficient mice and (2) oral administration of OVA with cholera toxin successfully primed T cells in B-cell-deficient mice. Thus, it was revealed that B cells are not required for both priming and tolerization of T cells to orally administered antigens. These results also contradict the supposed roles of PPs in mucosal immune responses.     

Pretreatment with minor histocompatibility antigens prevents the development of functional CTL helper cell activity

We have been studying the regulation of allogeneic cytotoxic cells (CTL) in vivo. CBA/J (H-2k, mls d) responder mice are unable to develop CTL after an allogeneic footpad immunization if they are pretreated i.p. with spleen cells from either C3H/HeN (H-2k, mls c) or B10.BR (H-2k, mls b) mice. These mouse strain combinations are H-2 compatible but differ at the Mls and other minor histocompatibility loci. We reported that this state of CTL unresponsiveness is specific and that the allogeneic cells used for footpad immunization and the pretreatment strain must share both minor antigens and part of the MHC. In this paper, we describe some of the characteristic features of this CTL unresponsiveness. The CBA host plays an active role and appears to down-regulate its subsequent response against minor antigens after the initial pretreatment. This statement is based on the following: 1) The inhibition of in vivo CTL generation can be achieved by injecting F1 or irradiated C3H cells, i.e., under conditions where GVHD was not a factor; and 2) the state of unresponsiveness is abolished by host treatment with cyclophosphamide. In addition, we demonstrate that the lack of CTL development in pretreated responder animals is the result of impaired helper cell activity. Draining LNC from unresponsive mice can become functionally cytolytic if cultured in a Con A-activated spleen cell supernatant. However, normal CTL responses were not restored after adult thymectomy or splenectomy. Thus, the state of CTL inhibition that is induced by the minor antigen pretreatment is the result of a host-mediated regulatory circuit.     

Responses against single minor histocompatibility antigens. II. Analysis of cloned helper T cells

We have utilized a clonal approach to investigate functional and immunogenetic characteristics of T cells responsive to the miHA H-1.3. Our data prove the existence of H-2 I region-restricted helper T cells that are specific for H-1.3, or for determinants encoded by closely linked loci. These conclusions are based upon the following observations: 1) the cloned Th are driven to proliferate only by H-2 I-compatible stimulator cells that bear the appropriate (H-1.3) miHA; 2) antigenic stimulation causes the cloned Th to secrete lymphokine with high levels of IL 2-like activity; and 3) they are not specifically lytic for relevant target cells. These cloned Th were isolated at a rather low frequency from an H-1.3-immune MLC population compared with the observed frequency of H-1.3-specific Tc that proliferate autonomously after antigen stimulation; i.e., HITc isolated from the same MLC. They were, however, capable of promoting the clonal expansion of an H-1.3-specific HITc, suggesting that Th can collaborate with HITc in an anti-miHA response.     

Stimulation of alloreactive cytotoxic T cells by paternal major histocompatibility antigens during murine pregnancy

In an effort to elucidate T cell reactivity toward paternal major histocompatibility (MHC) antigens during pregnancy, the ability of pregnant mice to develop alloreactive cytotoxic T lymphocytes (CTL) was studied in individual multiparous females mated with MHC congeneic strains of B10 background. Spleen cells obtained from B10.BR females mated to allogeneic males manifested strikingly higher CTL than those from animals mated to syngeneic males or from virgins; syngeneically mated animals were equivalent to virgin controls in CTL responses. The augmented CTL response in allogeneic pregnancy was detected not only by stimulation with the paternal MHC antigens but also by an unrelated MHC haplotype. However, this augmentation was found only during pregnancy in that 2-5 days after the delivery the CTL activity in allopregnant animals returned to a level comparable to that of virgin controls. No suppressor cells were detected at this stage. These observations suggest that maternal T cells recognize MHC disparity with the fetus in some way during pregnancy. Anti-MHC antibodies, immunoglobulin (Ig) M, and IgGs of all subclasses were not detected in these animals throughout multiple pregnancies.     

Retrovirus-induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes

Retrovirus infection of murine fibroblasts was found to alter the expression of major histocompatibility complex (MHC) antigens. Fibroblasts infected with Moloney murine leukemia virus (M-MuLV) exhibited up to a 10-fold increase in cell surface expression of all three class I MHC antigens. Increases in MHC expression resulted in the increased susceptibility of M-MuLV-infected cells to lysis by allospecific cytotoxic T lymphocytes (CTL). M-MuLV appears to exert its effect at the genomic level, because mRNA specific for class I antigens, as well as beta 2-microglobulin, show a fourfold increase. Fibroblasts infected with the Moloney sarcoma virus (MSV):M-MuLV complex show no increase in MHC antigen expression or class I mRNA synthesis, suggesting that co-infection with MSV inhibits M-MuLV enhancement of MHC gene expression. Quantitative differences in class I antigen expression on virus-infected cells were also found to influence the susceptibility of infected cells to lysis by H-2-restricted, virus-specific CTL. Differential lysis of infected cells expressing varied levels of class I antigens by M-MuLV-specific bulk CTL populations and CTL clones suggests that individual clones may have different quantitative requirements for class I antigen expression. The MSV inhibition of MHC expression could be reversed by interferon-gamma. Treatment of MSV:M-MuLV-infected fibroblasts with interferon-gamma increased their susceptibility to lysis by both allogeneic and syngeneic CTL. The data suggest that interferon-gamma may function in the host's immune response to viral infections by enhancing MHC antigen expression, thereby increasing the susceptibility of virus-infected cells to lysis by H-2-restricted, virus-specific CTL.